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Vesicular stomatitis virus induces apoptosis primarily through Bak rather than Bax by
inactivating Mcl-1 and Bcl- XL
Alicia F. Pearce and Douglas S. Lyles*
Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem,
North Carolina 27157
Running title: VSV induces apoptosis primarily through Bak rather than Bax
Abstract word count: 220
Text word count: 5,682
*Corresponding author. Mailing address: Department of Biochemistry, Wake Forest University
School of Medicine, Winston-Salem, NC 27157. Phone: (336) 716-4237. Fax: (336) 716-7671.
E-mail: [email protected].
Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Virol. doi:10.1128/JVI.00436-09 JVI Accepts, published online ahead of print on 8 July 2009
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ABSTRACT1
Vesicular stomatitis virus (VSV) induces apoptosis via the mitochondrial pathway. The 2
mitochondrial pathway is regulated by the Bcl-2 family of proteins which consists of both pro- 3
and anti-apoptotic members. To determine the relative importance of the multidomain 4
proapoptotic Bcl-2 family members, Bak and Bax, HeLa cells were transfected with Bak and/or 5
Bax siRNA and subsequently infected with recombinant wild-type (rwt) VSV. Our results 6
showed that Bak is more important than Bax for the induction of apoptosis in this system. Bak is 7
regulated by two anti-apoptotic Bcl-2 proteins, Mcl-1, which is rapidly turned over, and Bcl-XL, 8
which is relatively stable. Inhibition of host gene expression by the VSV M protein resulted in 9
the degradation of Mcl-1, but not Bcl- XL. However, inactivation of both Mcl-1 and Bcl- XL was 10
required for cells to undergo apoptosis. While inactivation of Mcl-1 was due to inhibition of its 11
expression, inactivation of Bcl-XL indicates a role for one or more BH3-only Bcl-2 family 12
members. VSV-induced apoptosis was inhibited by transfection with siRNA against Bid, a BH3-13
only protein that is normally activated by cleavage by caspase-8, the initiator caspase associated 14
with the death receptor pathway. Similarly, treatment with an inhibitor of caspase-8 inhibited 15
VSV-induced apoptosis. These results indicate a role for cross-talk from the death receptor 16
pathway in the activation of the mitochondrial pathway by VSV. 17
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INTRODUCTION 19
The induction of cell death is a major mechanism by which many viruses cause disease in 20
the tissues they infect (23). In addition, the cytolytic activity of viruses has the potential for 21
therapeutic applications such as the development of oncolytic viruses for the treatment of cancer 22
(27). Vesicular stomatitis virus (VSV) is well studied as a prototype for negative-strand RNA 23
viruses, and is an exceptionally potent inducer of apoptosis in a wide variety of cell types (4, 20, 24
21). Due to its particularly rapid cytopathic effects, VSV is one of the major viruses being 25
developed as an oncolytic agent (27). VSV is capable of inducing apoptosis by activation of 26
multiple apoptotic pathways. It is important to determine how these pathways are activated and 27
the role they play in apoptosis induced by VSV in order to understand the virulence and 28
oncolytic activity of the virus as well as to provide a model by which other viruses can be 29
compared. 30
Previous work showed that wild-type (wt) VSV induces apoptosis via the mitochondrial 31
(intrinsic) pathway through the initiator caspase, caspase-9 (4, 19). This is due in part to the 32
inhibition of host gene expression by the VSV M protein (19). The inhibition of host gene 33
expression by M protein is the mechanism by which VSV inhibits the host antiviral response (2, 34
31) and leads to induction of apoptosis, similar to that induced by pharmacologic inhibitors of 35
host gene expression (19). Additionally, M protein mutants of VSV that are deficient in the 36
ability to inhibit new host gene expression are effective inducers of apoptosis (12, 13, 19, 20). 37
However, in contrast to wt VSV, induction of apoptosis by M protein mutant virus occurs 38
primarily via the extrinsic pathway through the initiator caspase, caspase-8 (12, 13). Infection 39
with M protein mutant VSV results in the expression of proapoptotic genes that are suppressed 40
during infection with wt VSV (12). Therefore, in the case of VSV with wt M protein, the 41
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induction of apoptosis is most likely mediated by proteins already present in the host cell. Since 42
it has previously been shown that wt VSV activates the intrinsic pathway, we focused on the Bcl-43
2 family of proteins to determine the role of Bcl-2 family members in apoptosis induced by wt 44
VSV. 45
Bcl-2 family proteins function to either suppress or promote mitochondrial outer 46
membrane permeablization, thereby regulating the release of proapoptotic factors into the 47
cytosol, such as cytochrome c, apoptosis inducing factor (AIF) and Smac/Diablo (5). Bcl-2 48
family proteins are subdivided into three groups depending on conservation of Bcl-2 homology 49
(BH) domains and function (reviewed in (8, 38)). The multidomain antiapoptotic Bcl-2 proteins 50
contain BH domains BH1 - BH4 and function to inhibit apoptosis by binding to proapoptotic 51
Bcl-2 family members. Members of this group include Bcl-2, Bcl-XL, Mcl-1, Bcl-w, and BFL-52
1/A1. The proapoptotic Bcl-2 proteins are comprised of two groups, the multidomain proteins 53
and the BH3-only proteins. Bax and Bak are the two main members of the multidomain group, 54
containing BH domains BH1-BH3. These proteins are primarily responsible for the 55
permeabilization of the mitochondrial outer membrane, if their activity is not suppressed by 56
antiapoptotic Bcl-2 family members. The BH3-only proteins contain only one Bcl-2 homology 57
domain (BH3) and include Bid, Bad, Bim, Puma, Noxa and Bik, among others. These proteins 58
function as upstream sensors of signaling pathways and convey to other Bcl-2 family proteins the 59
signals to initiate apoptosis. These death signals can be transmitted from the BH3-only proteins 60
by either binding to antiapoptotic proteins causing the release of Bak and Bax, or they may bind 61
to Bak and Bax thereby causing their activation (6). 62
The pathways leading to activation of Bak differ from those that activate Bax. 63
Interestingly, only two antiapoptotic Bcl-2 proteins, Mcl-1 and Bcl-XL, have been shown to 64
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interact with Bak, while Bax appears able to interact with all of the antiapoptotic proteins with 65
the exception of Mcl-1 (7, 35). BH3-only proteins have strong binding affinities to the 66
antiapoptotic proteins, suggesting that their primary role may be to derepress Bak and Bax by 67
binding and inhibiting the antiapoptotic proteins (36). In addition, BH3-only proteins may play a 68
role in activation of Bak and Bax by binding and inducing an activated conformation (6, 34). For 69
some stimuli, such as the protein kinase inhibitor staurosporine, the topoisomerase II inhibitor 70
etoposide, and ultraviolet radiation, Bak and Bax appear to be redundant in that deletion of both 71
is required to render cells resistant to these agents (33). In contrast, Bak and Bax were non-72
redundant in the induction of apoptosis by Neisseria gonorrheae and cisplatin such that both 73
were required for apoptosis to occur (18). 74
In the experiments reported here, silencing of Bak or Bax expression with siRNA showed 75
that Bak is more important than Bax for the induction of apoptosis in HeLa cells infected with wt 76
VSV. Overexpression of both antiapoptotic Bcl-2 family proteins known to interact with Bak, 77
Mcl-1 and Bcl-XL, delayed the onset of apoptosis, while depletion of Mcl-1 or Bcl-XL by siRNA 78
transfection prior to infection increased the rate of apoptosis. Furthermore, M protein inhibition 79
of new host gene expression led to the depletion of Mcl-1 enabling the rapid activation of 80
apoptosis. However, inhibition of Bcl-XL was also required for the initiation of apoptosis, 81
indicating a role for one or more BH3-only proteins. Bid, a BH3-only protein that is normally 82
activated by cleavage by caspase-8, was shown to be important for induction of apoptosis by 83
VSV. Likewise, treatment with an inhibitor of caspase-8 inhibited VSV-induced apoptosis. 84
These results indicate a role for cross-talk from the death receptor pathway in the activation of 85
the mitochondrial pathway by VSV. 86
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MATERIALS AND METHODS 87
Cell lines and viruses. HeLa cells were cultured in Dulbecco’s modified Eagle medium 88
supplemented with 7% fetal bovine serum (FBS). The recombinant viruses rwt and rM51R-M 89
were isolated from cDNA clones and grown as previously described (20). Infections were 90
carried out at a multiplicity of infection of 10 PFU per cell. Stably transfected cell lines were 91
generated by transfecting HeLa cells with the h-Mcl-1 pcDNA3 plasmid or the h-Mcl-1-S159A 92
pc3DNA plasmid (gifts of Ulrich Maurer (24)), h-Bcl-XL pcDNA3 (gift of John Wilkinson) or 93
pcDNA3 plasmid for empty vector (EV) cells as previously described (19). Stably transfected 94
cells were cultured in Dulbecco’s modified Eagle medium supplemented with 7% FBS and 200 95
µg/ml of G418. 96
RNA Interference. For silencing experiments, HeLa cells were transiently transfected 97
with Bak siRNA (h), Bax siRNA (h), Bcl-XS/L (h), Control siRNA-A (Santa Cruz Biotechnology) 98
or On-Targetplus Duplex J-004501-17 Human Mcl-1 (Dharmacon) using TransIT-siQuest 99
transfection reagent (Mirus Bio Corporation). Briefly, HeLa cells were grown to approximately 100
75% confluence in 6-well plates and just before transfection, 1250 µL of fresh growth medium 101
was added to each well. For each well, a mixture of 250 µL Optimem media, 2 µL TransIT-102
siQuest transfection reagent and 25nM siRNA was incubated for 20min before adding to the 103
plate. After 24 hrs, cells were split into 96-well plates at a density of 8 x 103 for cell viability 104
and caspase-3-like activity assays, 6-well plates for cell lysates to confirm silencing and 6- or 24-105
well plates for time-lapse microscopy experiments. At 48 hrs posttransfection, cells were 106
infected with rwt virus. 107
Immunoblot analysis. HeLa cells or siRNA transfected HeLa cells were grown to 108
approximately 75% confluence in 6-well dishes and infected with recombinant viruses. At the 109
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indicated times postinfection, cell lysates were generated and analyzed by SDS-PAGE and 110
immunoblotting as previously described (13). The antibodies used in this study were obtained as 111
follows: Bak and Mcl-1 were from Santa Cruz Biotechnology, Bax and Bcl-XL were from Cell 112
Signaling Technologies and β-actin was from Sigma-Aldrich Incorporated. Protein band 113
intensities were quantitated by scanning and analysis with Quantity One software (Bio-Rad). 114
Time-lapse microscopy. siRNA transfected HeLa cells (described above) were grown to 115
approximately 50% confluence in 6- or 24-well dishes and infected with rwt virus. Time-lapse 116
microscopy was performed on a Zeiss Axiovert S200 as previously described (13). Fields 117
containing 40 – 80 cells were selected for analysis, and images of the same fields were captured 118
at 15-min intervals for 16 hrs. The time of entry of each cell into apoptosis was determined by 119
the time of onset of membrane blebbing that was followed by other morphological changes that 120
are characteristic of apoptosis as described in detail in (20). Data from three separate 121
experiments, including 120 – 200 total cells were analyzed and evaluated for statistical 122
significance by the log-rank test. 123
Cell viability assay. Cell viability was assessed by MTT assay, according to 124
manufacturer's instructions (Roche Molecular Biochemicals, Indianapolis, IN, USA). siRNA 125
transfected cells were seeded in 96-well plates as described above. Cell viability was evaluated 126
as described previously and survival estimated relative to untreated controls (19). 127
Caspase-3 activity assay. siRNA transfected cells were grown to approximately 75% 128
confluence as described above. Cells were either infected with rwt virus or treated with 129
staurosporine (SSP) (1 µg/ml; Cell Signaling Technologies) as a positive control for the 130
activation of the mitochondrial pathway. Duplicate wells were lysed and caspase-3 activation 131
was determined with a fluorogenic substrate for caspase-3 (DVED-AFC; R&D Systems, Inc.) as 132
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previously described (13). Prior to addition of the fluorogenic substrate, 10 µL were removed 133
and used to determine the protein concentration by Bio-Rad DC Protein Assay (Bio-Rad 134
Laboratories). 135
Statistical analysis. A paired Student’s t test was used to compare the significances of 136
differences between groups at individual time points for experiments containing two 137
experimental groups. Analysis of variance with Dunnett’s pairwise comparison with a control 138
was used for experiments containing more than two experimental groups using SigmaStat 139
software (Systat Software, Inc.). Time lapse microscopy data were analyzed for statistical 140
significance by the log-rank test (http://bioinf.wehi.edu.au/software/russell/logrank/index.html). 141
A P value of <0.05 was considered statistically significant. 142
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RESULTS 143
VSV induces apoptosis more slowly in HeLa cells lacking Bak than those lacking Bax. 144
Wild-type strains of VSV induce apoptosis primarily via the mitochondrial pathway (4, 19). 145
Since the mitochondrial pathway is regulated by the Bcl-2 family of proteins, we determined 146
which Bcl-2 family members play a role in VSV-induced apoptosis. Proapoptotic proteins Bax 147
and Bak in their active form are the main proteins responsible for the destabilization of the 148
mitochondrial membrane. Bak and Bax have been shown to be redundant in some systems (33), 149
while in other systems one is more important than the other (18). To determine the relative 150
importance of Bak and Bax in VSV-induced apoptosis, HeLa cells were transfected with siRNA 151
against either Bak, Bax, or both, or with non-targeting siRNA as a control. At 24, 48 and 72 hrs 152
post-transfection, cell lysates were prepared and analyzed by SDS-PAGE and immunoblotting. 153
Figure 1A shows a representative immunoblot for the silencing of Bak or Bax with analysis of 154
actin as a loading control. Levels of Bak and Bax were similarly low at all times post-155
transfection. Cells were infected with virus at 48 hrs post-transfection for all subsequent 156
experiments. In the experiments shown in Fig. 1B, cells were infected with a recombinant wild-157
type (rwt) strain of VSV and imaged using time-lapse microscopy. The percentages of cells 158
entering apoptosis were determined as a function of time postinfection by the time of onset of 159
apoptotic membrane blebbing. The graph shows the average of 3 experiments. Rwt virus rapidly 160
induced apoptosis in the control non-targeting siRNA-transfected cells (open circles, open 161
triangles) with over 75% of cells undergoing apoptosis by 16 hrs postinfection. A similar 162
timecourse was observed for the Bax siRNA cells (closed squares). However, the onset of 163
apoptotic membrane blebbing was delayed in both the Bak siRNA cells (closed circles) and the 164
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Bak plus Bax siRNA cells (closed triangles) with less than 30% of cells undergoing apoptosis by 165
16 hrs postinfection. 166
The delay in the induction of apoptosis in Bak siRNA cells was confirmed by an analysis 167
of caspase-3-like activity in cell lysates using a fluorogenic substrate (Fig. 1C) and by analysis of 168
cell viability using the MTT assay (Fig. 1D) which measures metabolically active cells. For the 169
caspase-3 assay, cells treated with staurosporine (SSP) were used as a positive control. Bax and 170
Bak siRNA cells both had caspase-3-like activity when treated with SSP comparable to that of 171
control non-targeting siRNA cells (data not shown). Caspase-3-like activity is presented in Fig. 172
1C as a percent of maximal SSP activation. For the MTT assay, data are expressed as a percent 173
of mock infected cells (Fig. 1D). Control siRNA cells as well as Bax siRNA had high levels of 174
caspase-3-like activity and reduced viability by 16 hrs postinfection while the Bak siRNA cells 175
and Bak plus Bax siRNA cells had significantly less active caspase-3 and higher cell viability 176
than control siRNA cells at 10, 12, and 16 hrs postinfection (p<.05)(Figs. 1C,D). Collectively, 177
the data in Figure 1 (B, C, D) show that silencing of Bak expression slows the rate of apoptosis, 178
while silencing of Bax expression does not. 179
Mcl-1 levels are reduced in VSV-infected HeLa cells while Bcl-XL levels remain 180
unchanged. In healthy cells, Bak is kept in an inactive state by interaction with anti-apoptotic 181
Bcl-2 family members. Only two anti-apoptotic family members, Bcl-XL and Mcl-1, have been 182
found to interact directly with Bak. Bcl-XL is a relatively stable protein with a half-life of over 183
24 hrs (1). However, Mcl-1 protein and mRNA have been shown to have a rapid turnover rate in 184
many cell types (1, 26). For example, the half-life of Mcl-1 in cycloheximide-treated HeLa cells 185
is <1 hr (1), which we have confirmed (data not shown). VSV inhibits new host gene expression 186
due to the ability of M protein to inhibit host transcription, nuclear-cytoplasmic transport and 187
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translation (23). We hypothesized that Mcl-1 levels would decay rapidly in VSV-infected cells 188
allowing for the activation of Bak and subsequent apoptosis. To test this hypothesis, HeLa cells 189
were infected with rwt virus and at different times postinfection cell lysates were prepared and 190
analyzed for Mcl-1 by SDS-PAGE and immunoblotting (Fig. 2A). There was a visible reduction 191
in the levels of Mcl-1 in rwt virus-infected HeLa cells by 8 hrs postinfection with levels below 192
the level of detection by 24 hrs postinfection. The timing of reduction in Mcl-1 levels was 193
consistent with the induction of apoptosis (Fig. 1) and with the inhibition of host gene expression 194
(2). To confirm that the loss of Mcl-1 was due to the shut-off of new host gene expression, HeLa 195
cells were infected with a recombinant M protein mutant virus that is isogenic with rwt virus 196
except for a point mutation in the M protein, which renders the virus defective in the ability to 197
inhibit host gene expression (rM51R-M virus). Figure 2A shows quantification of Mcl-1, 198
expressed as a ratio to actin and normalized to mock infected controls. There was a significant 199
reduction in the levels of Mcl-1 in rwt virus-infected cells (closed squares) by 8 hrs postinfection 200
with levels continuing to decline throughout the 24 hr timecourse. In contrast, Mcl-1 levels were 201
increased by 4 hrs postinfection and were similar to mock-infected cells (open circles) for the 202
remainder of the timecourse in rM51R-M virus-infected cells (open squares). A similar 203
immunoblot analysis of Bcl-XL levels showed that, in contrast to Mcl-1, there were no 204
substantial changes in the level of Bcl-XL in rwt virus-infected cells at any time postinfection 205
when compared to the level in mock-infected cells (Fig 2B). These data support the hypothesis 206
that loss of Mcl-1 is due to its rapid turnover combined with the inhibition of host gene 207
expression by wt VSV. 208
Apoptosis is regulated by levels of both propapoptotic and antiapoptotic Bcl-2 proteins, 209
therefore we analyzed the levels of proapoptotic proteins Bak and Bax by immunoblots, as well 210
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(Fig. 3). There were no significant changes in the level of Bak at any time postinfection (Fig. 211
3A). Levels of Bax began to decline at 12 hrs postinfection and were significantly decreased by 212
24 hrs postinfection (Fig. 3B), likely due to normal turnover following virus-induced inhibition 213
of its synthesis. By contrast, levels of Bak can remain unaffected for long periods of time (>16 214
hrs) following inhibition of its synthesis (17). The observation that levels of Bak did not decline 215
during infection is consistent with the idea that loss of Mcl-1 contributes to the activation of Bak. 216
Since Bax levels do not have an effect on the rate of apoptosis in rwt virus-infected cells (Fig. 1), 217
the decrease in the level of Bax at late times postinfection is not likely to be important in VSV-218
induced apoptosis in HeLa cells. 219
Apoptosis induced by VSV infection is delayed in HeLa cells that overexpress Mcl-1 or 220
Bcl-XL. To determine whether the rapid decrease in the level of Mcl-1 contributes to the 221
induction of apoptosis in rwt virus infected cells, HeLa cells that stably overexpress Mcl-1 were 222
generated. Due to the short half-life of Mcl-1, we also generated cell lines that express a mutant 223
Mcl-1, S159A. This is a phosphorylation site mutant, which exhibits enhanced stability and 224
confers increased protection from apoptosis compared to wt Mcl-1 (24). Two clonal cell lines 225
were generated for both the wt Mcl-1 and the mutant Mcl-1 and are both shown to indicate 226
differences are not due to clonal variation. The cell lines overexpress Mcl-1 compared to an 227
empty vector (EV) control cell line as determined by immunoblots (Fig. 4A). During infection 228
with rwt virus, the levels of Mcl-1 declined in HeLa-wt Mcl-1 cells with a time course similar to 229
that in HeLa-mutant Mcl-1 cells, as determined by quantification of immunoblots similar to 230
those in Fig. 4A (Fig. 4B). Thus the greater stability of the mutant Mcl-1 was not sufficient to 231
delay its decline in rwt virus-infected cells. Nonetheless, the levels of Mcl-1 in both HeLa-wt 232
Mcl-1 and HeLa-mutant Mcl-1 cells were higher than in HeLa-EV control cells throughout the 233
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first 12 hr postinfection. To determine if the increase in Mcl-1 levels delayed the induction of 234
apoptosis by rwt virus, HeLa-wt Mcl-1, HeLa mutant Mcl-1 and HeLa-EV cells were infected 235
with rwt virus, and cells entering apoptosis were quantitated using time-lapse microscopy (Fig. 236
4C). HeLa-EV cells entered apoptosis with a timecourse similar to untransfected HeLa cells 237
(Fig. 1). Compared to control cells, there was approximately a 1 hr delay in the time it took for 238
50% of cells to enter apoptosis for the two wt Mcl-1-overexpressing cell lines, and a 2 hr delay 239
for the two mutant Mcl-1 overexpressing cell lines. The effects of overexpression of Mcl-1 on 240
virus-induced apoptosis were also determined by assaying caspase-3-like activity. In contrast to 241
the relatively modest change in the timecourse of onset of membrane blebbing, the level of 242
caspase-3 activated in HeLa-Mcl-1 cells was dramatically reduced compared to that activated in 243
HeLa-EV cells (Fig. 4D). This difference in magnitude of the effects of Mcl-1 overexpression 244
may be due to how little caspase-3 activation is required to cause HeLa cells to undergo 245
morphological changes associated with apoptosis. Alternatively, there may be caspase-3-246
independent mechanisms that lead to the morphological changes assayed in Fig. 4B. From these 247
data we concluded that Mcl-1 was able to marginally inhibit the onset of the morphological 248
changes accompanying apoptosis in rwt virus-infected HeLa cells, but there was a substantial 249
reduction in activation of caspase-3-like activity. 250
Since there was a decrease in the rate of apoptosis in cells transfected with Mcl-1 despite 251
the rapid turnover, we hypothesized that overexpression of a more stable antiapoptotic Bcl-2 252
family member, i.e., Bcl-XL, would have a more dramatic effect on the induction of apoptosis 253
compared to overexpression of Mcl-1. To determine the effects of Bcl-XL overexpression, a 254
HeLa cell line that stably overexpressed Bcl-XL was generated. An immunoblot demonstrating 255
the overexpression of Bcl-XL is shown in Fig. 5A. The induction of apoptosis in HeLa-Bcl- XL 256
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cells and HeLa-EV cells infected with rwt virus was quantified by time-lapse microscopy (Fig. 257
5B) and by assay of caspase-3-like activity (Fig. 5C). Rwt virus rapidly induced apoptosis in the 258
control, empty vector cells (open circles) with approximately 90% of cells undergoing apoptosis 259
by 16 hrs postinfection, while the only 20% of Bcl-XL transfected cells (closed squares) 260
underwent apoptosis by the same time (Fig. 5B). Similar results were obtained for the caspase-3 261
assay in which caspase-3-like activity was almost completely abrogated in the Bcl-XL transfected 262
cells (Fig. 5C). Both assays showed that overexpression of the more stable antiapoptotic protein, 263
Bcl-XL caused a dramatic reduction in the rate of induction of apoptosis in rwt virus-infected 264
cells compared to overexpression of Mcl-1. 265
Apoptosis induced by VSV infection is accelerated in HeLa cells transfected with Mcl-1 266
or Bcl-XL siRNA. The data in Figs. 2-5 indicate that the rapid induction of apoptosis by rwt virus 267
in HeLa cells is due in part to the depletion of Mcl-1. SiRNA silencing experiments were used 268
to determine whether loss of Mcl-1 or Bcl-XL or both was sufficient to cause cells to undergo 269
apoptosis. Fig. 6A is a representative immunoblot showing the siRNA silencing of Mcl-1 and 270
Bcl-XL. There was no significant reduction in cell viability of Mcl-1 or Bcl-XL siRNA cells at 271
either 24 or 48 hrs post-transfection determined by MTT asay (Fig. 6B). However, the combined 272
silencing of Mcl-1 and Bcl-XL decreased cell viability to approximately 40% of control at both 273
timepoints. Thus, silencing of either Mcl-1 or Bcl- XL alone was not sufficient to induce 274
apoptosis, while silencing of both was sufficient to induce cell death in the absence of a viral 275
infection. These results suggest that the loss of Mcl-1 alone in VSV-infected cells is not 276
sufficient to induce apoptosis, and suggest that inactivation of Bcl-XL is also required. 277
To test whether silencing of Mcl-1 or Bcl-XL would accelerate virus-induced apoptosis, 278
HeLa cells were transfected with either Bcl-XL, Mcl-1, or non-targeting siRNA for 48 hrs and 279
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then infected with rwt virus. Silencing of Mcl-1 led to a slightly faster induction of apoptosis 280
than in control siRNA cells as measured by time-lapse microscopy (open circles versus closed 281
triangles, Fig. 7A). However, silencing of Bcl-XL (closed squares) dramatically increased the 282
rate of virus-induced apoptosis. Similar results were obtained by assaying caspase-3-like activity 283
and cell viability (Fig. 7B and 7C). There was no significant difference in the amount of virus-284
induced caspase-3-like activity or reduction in cell viability in Mcl-1 siRNA cells as compared to 285
control siRNA cells throughout the time course (Fig. 7B). In contrast, virus infection activated 286
significantly more caspase-3 and reduced cell viability to a significantly greater extent in Bcl-XL 287
siRNA cells than in control siRNA cells. These data are consistent with the idea that Mcl-1 is 288
degraded quickly upon infection with VSV, therefore removing Mcl-1 prior to infection does not 289
increase the rate of apoptosis. However, inhibition of Bcl-XL requires the activation of one or 290
more BH3 only proteins, so removing Bcl-XL prior to infection significantly increases the rate of 291
apoptosis, presumably by reducing the time required for inactivation of antiapoptotic proteins by 292
activated BH-3 only proteins. 293
Apoptosis induced by VSV infection is delayed in HeLa cells transfected with Bid siRNA 294
or treated with caspase-8 inhibitor. The experiments in Fig. 7 raise the question of which BH3-295
only protein(s) are involved in the inactivation of Bcl-XL. Our previous experiments have shown 296
that VSV infection activates elements of the death receptor pathway as well as the mitochondrial 297
pathway (12, 13, 19). Cross-talk between the death receptor pathway and the mitochondrial 298
pathway is mediated primarily by the BH3-only protein Bid, which is cleaved to its active form 299
(truncated Bid or t-Bid) by caspases-8 and -10 (28, 39). We tested the hypothesis that induction 300
of apoptosis by VSV involves activation of Bid by analysis of Bid cleavage by immunoblots and 301
by silencing Bid expression by transfection with siRNA. HeLa cells were infected with rwt virus, 302
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and Bid cleavage was analyzed by immunoblots (Fig. 8A), using an antibody raised against the 303
N-terminus, which is present in full-length Bid, but not t-Bid. Quantification of multiple 304
experiments showed that more than 50% of Bid was cleaved by 8 hr postinfection, which 305
generally precedes the onset of the morphological changes associated with the induction of 306
apoptosis (e.g., Figs. 1 and 4), and thus is a very early event in the induction of apoptosis by 307
VSV. Also shown in Fig. 8A, the rapid cleavage of full-length Bid was prevented by treatment of 308
cells with a caspase-8 inhibitor, IETD-fmk (note the difference in time scale), indicating that 309
disappearance of full-length Bid was due to caspase cleavage and not due to normal turnover, 310
following inhibition of its synthesis following VSV infection. 311
To determine whether silencing of Bid would decrease the rate of VSV-induced 312
apoptosis, HeLa cells were transfected with either Bid or non-targeting siRNA for 48 hrs and 313
then infected with rwt virus. An immunoblot of transfected cells is shown in Fig. 8B, which 314
indicated that Bid expression was reduced to 24% of control prior to infection. Most of the Bid 315
siRNA cells entered apoptosis more slowly than control siRNA cells as measured by time-lapse 316
microscopy (open circles versus closed squares, Fig. 8B). Similar results were obtained by 317
assaying caspase-3-like activity and cell viability (Fig. 8C and 8D). These data are consistent 318
with the idea that activation of Bid during VSV infection leads to the inactivation of Bcl-XL 319
and/or the activation of Bak. 320
Since Bid is cleaved primarily by caspase-8, the experiments in Fig. 8 were extended by 321
treating HeLa cells with a caspase-8 inhibitor to determine whether the rate of VSV-induced 322
apoptosis was descreased. HeLa cells were preincubated with either caspase-8 inhibitor (Z-323
IETD-fmk), pan-caspase inhibitor (Z-VAD-fmk), or no inhibitor for 2 hrs prior to infection with 324
rwt virus. The inhibitors were present in the media throughout the timecourse of the experiment. 325
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Cell viability was analyzed at 12, 16, and 24 hrs postinfection by MTT assay. Similar treatment 326
with IETD-fmk of transfected cells that express M protein in the absence of other viral 327
components did not inhibit apoptosis (19). However, in the case of virus-infected HeLa cells, 328
caspase-8 inhibitor delayed cell death to an extent similar to that of pan-caspase inhibitor (Fig. 329
9A). Interestingly, even in the presence of a pan-caspase inhibitor, VSV was able to induce cell 330
death, suggesting that caspase-independent pathways may also be activated. These results 331
together with those in Fig. 8 indicate that elements of the death receptor as well as mitochondrial 332
pathway are important for VSV-induced apoptosis and that crosstalk between the two pathways 333
occurs via caspase-8 cleavage of Bid. This idea was further supported by infecting HeLa-Bcl-XL 334
cells with rwt virus in the presence of caspase-8 inhibitor or pan caspase inhibitor (Fig. 9B). In 335
the absence of inhibitors, the survival of HeLa-Bcl-XL cells was dramatically prolonged 336
compared to control HeLa cells (compare the time scales in Fig. 9A versus 9B). Death of HeLa-337
Bcl-XL cells was delayed slightly in the presence of caspase inhibitors, but the differences in cell 338
viability were not statistically significant at most time points. These data indicate that the 339
contribution of the death receptor pathway to induction of apoptosis by rwt virus is primarily 340
through cross-talk with the mitochondrial pathway. 341
342
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DISCUSSION 343
Cell death via the intrinsic pathway depends on the multidomain proapoptotic proteins 344
Bak and Bax (22) which function to permeabilize the outer mitochondrial membrane. In some 345
systems, Bak and Bax have been shown to be functionally redundant. Studies utilizing 346
knockout MEFs showed that MEFs deficient in either Bak or Bax alone underwent apoptosis 347
induced by multiple apoptotic stimuli. Conversely, deficiency in both Bak and Bax conferred 348
resistance to these apoptotic stimuli (33). These results indicate that there is functional 349
redundancy for Bak and Bax in MEFs. Subsequently, other groups have reported similar 350
functional overlap in different cell types (9, 15, 33) giving rise to the idea that Bak and Bax are 351
redundant regulators of intrinsic mitochondrial apoptosis and that inactivation of both is 352
necessary to abrogate cell death. The results presented here contrast with this view by showing 353
that Bak plays a more significant role than Bax in the induction of apoptosis in VSV infected 354
cells (Fig. 1). 355
Lack of functional redundancy of Bak and Bax is not surprising given the differences 356
between them. Inactive Bax is a latent monomer in the cytosol (16, 37). Once activated by 357
apoptotic stimuli, Bax undergoes conformational changes causing altered epitope availability, the 358
formation of homodimers and oligomers, and translocation to the mitochondrial membrane (8). 359
In contrast, Bak resides in a membrane-bound protein complex (14), which suggests that Bak and 360
Bax are activated by different mechanisms. In fact, recent evidence has shown that some 361
apoptotic stimuli, such as staurosporine, actinomycin D, TRAIL and overexpression of Puma 362
activate Bak preferentially, and that Bak can lead to the release of cytochrome c in the absence of 363
Bax activation (25). Our observation that, in the context of a VSV infection, Bak appears to 364
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play a greater role in the induction of apoptosis than Bax is consistent with these more recent 365
results. 366
Bak and Bax are usually constitutively expressed at relatively constant levels and are 367
mainly regulated post-translationally by other Bcl-2 proteins. Antiapoptotic proteins, such as 368
Bcl-2, Mcl-1 and Bcl-XL, keep Bax and Bak from permeabilizing the outer mitochondrial 369
membrane. One mechanism by which they accomplish this is by binding to BH3-only proteins 370
and inhibiting their activity. This is the likely mechanism by which overexpression of Bcl-2 371
inhibits induction of apoptosis by VSV (10, 19), since Bcl-2 is not known to bind directly to Bak. 372
Since Bax exists as a monomer in the cytosol, there is no repressor bound to Bax in healthy cells 373
(8). However, once apoptotic signals from BH3 only proteins have induced a conformational 374
change in Bax, it translocates to the mitochondrial membrane where Bax can be held in check by 375
Bcl-2, Bcl-B and Bcl-XL (40). Bak, constitutively present in membranes, has been shown to be 376
kept inactive by interaction with multidomain antiapoptotic Bcl-2 family members Mcl-1 and 377
Bcl-XL in organelle membranes (7, 35). Mcl-1 normally has a rapid turnover rate and is 378
degraded by the ubiquitin-proteosome pathway (1, 26). Therefore, we hypothesized that 379
inhibition of new host gene expression by the VSV M protein induces apoptosis due to the loss 380
of Mcl-1, since the protein cannot be replaced in VSV-infected cells once it has been degraded. 381
This hypothesis was supported by the timing of the reduction of Mcl-1 protein levels during 382
VSV infection which coincides with the induction of apoptosis (Fig. 2). While this manuscript 383
was in revision, similar results were published showing degradation of Mcl-1 following VSV 384
infection in other cell types (29). However, depletion of Mcl-1 alone is not sufficient to induce 385
apoptosis (Fig. 6) indicating that inactivation of Bcl-XL is also required for cells to enter 386
apoptosis. 387
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Inactivation of Bcl-XL in VSV-infected cells is likely due to activation of one or more 388
BH3-only proteins. Many BH3-only proteins have been described. However, M protein-389
mediated shut-off of new host gene expression would likely inhibit the expression of BH3-only 390
proteins that must be newly synthesized, which suggested that BH3-only proteins that are post-391
translationally regulated were the most likely candidates. Our previous research has shown that 392
the death receptor pathway is the principal pathway for induction of apoptosis by M protein 393
mutant VSV, rM51R-M virus (12, 13). This led to our hypothesis that the death receptor pathway 394
might also be activated by VSV with wt M protein, and that BH3-only protein Bid, which is 395
activated by proteolytic cleavage by caspase-8, was involved in Bcl-XL inactivation and Bak 396
activation. This hypothesis was supported by the significant delay in VSV induced apoptosis in 397
both Bid siRNA cells (Fig. 8) as well as in the presence of capase-8 inhibitor (Fig. 9). These 398
results suggest that both the intrinsic and extrinsic pathways are important for apoptosis induced 399
by VSV with wt M protein, and that crosstalk between the two pathways occurs via caspase-8 400
cleavage of Bid. The key elements of this pathway are summarized in Fig. 10. 401
The involvement of the death receptor pathway in the induction of apoptosis by rwt virus 402
contrasts with previous results from our laboratory on the induction of apoptosis by expression of 403
wt M protein in transfected cells in the absence of other viral components (19). Those 404
experiments showed that expression of M protein induced apoptosis through the mitochondrial 405
pathway, similar to pharmacologic inhibitors of host gene expression, and that capsase-8 406
inhibitor did not inhibit M protein-induced apoptosis. Combining those results with the results 407
presented here suggests that the inhibition of host gene expression by M protein may induce 408
activation of other BH3-only proteins besides Bid, which are independent of caspase-8 and the 409
death receptor pathway (dashed lines in Fig. 10). The presence of such an additional pathway 410
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may account for the fact that caspase inhibitors do not completely prevent cell death in HeLa 411
cells (Fig. 9), as well as other reports indicating that inhibition of caspase-8 does not completely 412
prevent VSV-induced apoptosis (3, 10, 11). The inability of caspase-8 inhibitor to completely 413
prevent cell death could also be due to the activity of other caspases that are coupled to the death 414
receptor pathway, such as caspase-10 (32). 415
The results presented here provide new insight into why different cell types vary in their 416
sensitivity to induction of apoptosis by VSV with wt versus mutant M protein (12, 13, 19, 20). 417
Cells that depend on Mcl-1 to keep Bak inactive will be particularly sensitive to viruses with wt 418
M protein, which inhibits host gene expression leading to Mcl-1 degradation. In contrast, some 419
cells are more sensitive to viruses encoding the M51R mutant M protein (13, 20). In these cells, 420
the expression of new proapoptotic gene products is required for the rapid induction of apoptosis, 421
and apoptosis occurs primarily through the death receptor pathway (12, 13). Even though the 422
mitochondrial pathway is activated, induction of apoptosis in these cells is independent of the 423
mitochondrial pathway. In addition, there are cells in which the induction of apoptosis by M 424
protein mutant virus is dependent on the mitochondrial pathway, presumably through cross-talk 425
with the death receptor pathway (30). The differences in requirement for activation of the 426
mitochondrial pathway by M protein mutant VSV is similar to the classification of cell types into 427
type I (mitochondria-independent) or type II (mitochondria-dependent) based on their response 428
to death ligands such as Fas (28). The results presented here together with previous results 429
suggest that VSV is a particularly potent inducer of apoptosis because it activates multiple 430
apoptotic pathways, whose expression varies considerably among cell types. 431
432
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ACKNOWLEDGEMENTS 433
We thank Margie McKenzie for performing the Bid immunoblots and Drs. Maryam 434
Ahmed, David Ornelles and John Wilkinson for helpful advice and comments on the manuscript. 435
We also thank Ulrich Maurer and John Wilkinson for kindly providing plasmids. This work was 436
supported by NIH grants R01-AI32983 and R01-AI52304. 437
438
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FIGURE LEGENDS 563 564
Figure 1: rwt virus induces apoptosis more slowly in HeLa cells lacking Bak than those lacking 565
Bax (A) HeLa cells were transfected with 25nM Bak, Bax or non-targeting siRNA as a control. 566
At the indicated times posttransfection, cell lysates were analyzed by immunoblotting with 567
antibodies for Bak, Bax and actin as a loading control. Cells transfected with Bak or Bax siRNA 568
were analyzed in duplicate. (B) HeLa cells were transfected with 25nM Bak siRNA (closed 569
circles), Bax siRNA (closed squares), Bak and Bax siRNA (closed triangles), 25nM non-570
targeting siRNA (open circle) as a control for single transfection, 50nM non-targeting siRNA 571
(open triangle) as a control for double transfection. At 48 hrs post-transfection, cells were 572
infected with rwt virus and analyzed by time-lapse microscopy. The cumulative percentage of 573
cells entering apoptosis was determined as the number of cells that underwent apoptotic 574
membrane blebbing and is plotted as a function of time postinfection. The data represent the 575
average of three experiments, which collectively analyzed the time of onset of apoptosis in 120 -576
200 total cells. Bak siRNA and Bak+Bax siRNA data were significantly different from control 577
and Bax siRNA data (p < 0.05) by log-rank analysis. In subsequent assays only one control is 578
shown as the data is almost identical. (C) Caspase-3-like activity was assayed in HeLa cells 579
transfected for 48 hrs with the same siRNAs as above, then infected with rwt virus for the times 580
indicated. Cells were lysed, and caspase-3 activity in cell lysates was measured with a 581
fluorogenic substrate (DEVD-AFC). Data are expressed in arbitrary fluorescence units per 582
microgram of total protein, and normalized to the maximum value expressed by HeLa cells 583
incubated with staurosporine (as a positive control) for 12 hrs. The data represent the average 584
+/- standard deviation of three experiments. (D) Cell viability was analyzed with an MTT assay. 585
HeLa cells were transfected and infected as above. At the indicated times, MTT was added to 586
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each sample for 4 hrs at which point a solubilization solution was added. Samples were analyzed 587
with an ELISA plate reader. Cell viability was determined as a percentage of a transfected, 588
uninfected control. The data represent the average +/- standard deviation of three experiments ( 589
*, p< 0.05 compared to control). 590
591
Figure 2: Mcl-1 levels are reduced in rwt virus infected HeLa cells while Bcl-XL levels remain 592
unchanged. (A) At the indicated times postinfection with rwt virus, cell lysates were analyzed 593
by immunoblotting with antibodies for Mcl-1. The graph shows quantitation of Mcl-1 expression 594
normalized to actin expression as percentages of the average ratio in mock infected cells. The 595
data represent the averages +/- standard deviation from three experiments for cells infected with 596
rwt virus (closed squares) or rM51R-M virus (open squares) or mock infected cells (open 597
circles). (B) Cells were analyzed for Bcl-XL expression as in (A). 598
599
Figure 3: Bak levels in rwt infected HeLa cells are not significantly different from mock 600
infected samples, while Bax levels decrease at late times postinfection. At the indicated times 601
postinfection with rwt virus, cell lysates were generated and analyzed by immunoblotting with 602
antibodies for Bak (A) and Bax (B). Representative gels are shown at the top of panels A and B 603
and the graphs show quantitation of Bak (A) or Bax (B) protein expression normalized to actin 604
protein expression and are shown as percentages of mock infected samples. The data represent 605
the averages +/- stand deviation from three experiments for cells infected with rwt virus (closed 606
squares) or mock-infected cells (open circles) . 607
608
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Figure 4: Apoptosis is delayed in Mcl-1 overexpressing cells infected with rwt virus. Clonal 609
HeLa cell lines that overexpress wildtype Mcl-1 (Mcl-1-13, Mcl-1-16), a mutant Mcl-1 (S159A-610
7, S159A-8) or an empty vector control were generated. (A) Cell lysates were analyzed by 611
immunoblotting using an antibody against Mcl-1. (B) Mcl-1-13 (open squares), Mcl-1-16 (closed 612
squares), S159A-7 (open triangles), S159A-8 (closed triangles) and empty vector cells (open 613
circles) were infected with rwt virus. The graph shows quantitation of Mcl-1 expression 614
normalized to actin expression as percentages of the average ratio in mock infected cells. The 615
data represent the averages +/- standard deviation from three experiments. (C) Mcl-1-13 (open 616
squares), Mcl-1-16 (closed squares), S159A-7 (open triangles), S159A-8 (closed triangles) and 617
empty vector cells (open circles) were infected with rwt virus. Cells entering apoptosis were 618
analyzed by time-lapse microscopy as described in the legend to Fig. 1. The data represent the 619
averages from three experiments, which collectively analyzed the time of onset of apoptosis in 620
120 -200 total cells. Data for Mcl-1-13 and Mcl-1-16 cells were significantly different from data 621
for control empty vector cells as well as data for S159A-7 and S159A-8 cells (p < 0.05) by log-622
rank analysis. (D) Mcl-1 overexpressing cells and empty vector cells were infected with rwt 623
virus for the times indicated and caspase-3-like activity was analyzed using a fluorogenic 624
substrate as described in the legend to Fig. 1. The data represent the averages +/- standard 625
deviation from three experiments. 626
627
Figure 5: Apoptosis is significantly delayed in Bcl-XL-overexpressing cells infected with rwt 628
virus. HeLa cells that overexpress Bcl-XL (Bcl-XL-3) or an empty vector control were generated. 629
(A) Cell lysates were generated and analyzed by immunoblotting using an antibody against Bcl-630
XL. (B) Bcl-XL-3 (closed squares) and empty vector cells (open circles) were infected with the 631
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rwt virus. Cells entering apoptosis were analyzed by time-lapse microscopy as described in the 632
legend to Fig. 1. The data represent the averages from three experiments, which collectively 633
analyzed the time of onset of apoptosis in 120 -200 total cells. Data for Bcl-XL-3 cells were 634
significantly different from data for control empty vector cells (p < 0.05) by log-rank analysis. 635
(C) Bcl-XL-3 and empty vector cells were infected with rwt virus for the times indicated and 636
caspase-3-like activity was analyzed using a fluorogenic substrate as described in the legend to 637
Fig. 1. The data represent the averages +/- standard deviation from three experiments ( *, p< 638
0.05 compared to empty vector control). 639
640
Figure 6: Simultaneous silencing of Bcl-XL and Mcl-1 by siRNA transfection induces cell death. 641
(A) HeLa cells were transfected in duplicate with 25nM Mcl-1, Mcl-1 and Bcl-XL or non-642
targeting (NT) siRNA as a control. At 24 hrs posttransfection, cell lysates were analyzed by 643
immunoblotting using antibodies against Mcl-1 and Bcl-XL. (B) HeLa cells were transfected 644
with Mcl-1, Bcl-XL, Mcl-1 and Bcl-XL or non-targeting siRNA as a control. Cell viability was 645
analyzed at 24 and 48 hrs posttransfection by MTT assay as described in the legend to Fig.1. 646
The data represent the average and standard deviation from three experiments. ( *, p< 0.05 647
compared to single silenced cells). 648
649
Figure 7: Apoptosis is induced significantly faster by rwt virus infection in Bcl-XL silenced 650
cells. (A) HeLa cells were transfected with Mcl-1 (closed triangles), Bcl-XL (closed squares) or 651
non-targeting siRNA (open circles) for 48 hrs and then infected with rwt virus. Cells entering 652
apoptosis were analyzed by timelapse microscopy as described in the legend to Fig. 1. The data 653
represent the averages from three experiments, which collectively analyzed the time of onset of 654
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apoptosis in 120 -200 total cells. Data for Mcl-1 siRNA cells were significantly different from 655
data for control siRNA cells as well as data for Bcl-XL siRNA cells (p < 0.05) by log-rank 656
analysis. (B) HeLa cells were transfected with Mcl-1, Bcl-XL or non-targeting siRNA for 48 hrs 657
and then infected with rwt virus for the times indicated and caspase-3-like activity was analyzed 658
using a fluorogenic substrate as described in the legend to Fig. 1. The data represent the 659
averages +/- standard deviation from three experiments. (C) HeLa cells were transfected with 660
Mcl-1, Bcl-XL, or non-targeting siRNA for 48 hrs and then infected with rwt virus. Cell viability 661
was analyzed at the indicated times by MTT assay as described in the legend to fig.1. The data 662
represent the average and standard deviation from three experiments ( *, p<0.05 compared to 663
control). 664
665
Figure 8: Apoptosis is significantly delayed in Bid silenced cells infected with rwt virus. (A) At 666
the indicated times postinfection with rwt virus, cell lysates were analyzed by immunoblotting 667
with antibodies for Bid. The graph shows quantitation of Bid expression as percentages of the 668
levels in mock infected cells. The data represent the averages +/- standard deviation from four 669
experiments. Shown on the right are results from HeLa cells incubated in the presence of 100 670
µM caspase-8 inhibitor (IETD-fmk) for 2 hrs prior to infection with rWT virus for the indicated 671
times. The inhibitor was present in the media throughout the timecourse. (B) HeLa cells were 672
transfected with Bid or non-targeting control siRNA or mock-transfected for 48 hrs and then 673
analyzed for Bid expression by immunoblots. Bid siRNA cells (closed squares) or control siRNA 674
cells (open circles) were infected with rWT virus. Cells entering apoptosis were analyzed by 675
timelapse microscopy as described in the legend to Fig. 1. The data represent the averages from 676
three experiments, which collectively analyzed the time of onset of apoptosis in 120 -200 total 677
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cells. Data for Bid siRNA cells were significantly different from data for control siRNA cells (p 678
< 0.05) by log-rank analysis. (C) HeLa cells were transfected with Bid or non-targeting siRNA 679
for 48 hrs and then infected with rWT virus for the times indicated and caspase-3-like activity 680
was analyzed using a fluorogenic substrate as described in the legend to Fig. 1. The data 681
represent the averages +/- standard deviation form three experiments. (D) HeLa cells were 682
transfected with Bid or non-targeting siRNA for 48 hrs and then infected with rWT virus. Cell 683
viability was analyzed at the indicated times by MTT assay as described in the legend to Fig. 1. 684
The data represent the average and standard deviation from three experiments. (*, p<.05 685
compared to control). 686
687
Figure 9: Apoptosis is significantly delayed in HeLa cells infected with rwt virus in the presence 688
of caspase-8 inhibitor. (A) HeLa cells or (B) HeLa cells that stably overexpress Bcl-XL were 689
incubated in the presence of 100 µM caspase-8 inhibitor, 100uM pan-caspase inhibitor or no 690
inhibitor for 2 hrs. Cells were then infected with rWT virus for the indicated times. The 691
inhibitors were present in the media throughout the timecourse. Cell viability was analyzed at 692
the indicated times by MTT assay as described in the legend to Fig. 1. The data represent the 693
average and standard deviation from three experiments. (*, p<.05 compared to control). 694
695
Figure 10: Major pathways involving Bcl-2 family members leading to apoptosis in HeLa cells 696
infected with VSV with wt M protein. 697
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